1. Field of the Invention
The present invention relates to a 4D biochip, methods for its use and methods for its production, wherein the 4D biochip provides high efficiency and high throughput testing.
2. Discussion of the Background
Large scale, multiple sample, parallel biochemistry assays, automated instruments, and system integration (instrument, databases and analytical tools) using the latest bioinformatics technologies are key factors for advancing the field of functional genomics. In recent years, DNA chip technology has been a focal point of genomic scientists and potential customers of genomics technology because of the ability of the DNA chip to assay a large number of genes in parallel. DNA chip technology can be used, for example, in gene expression assaying (parallel Northern blotting) to determine gene functions, in polymorphism detection and molecular marker genotyping (for example, SNP), to provide efficient genetic mapping, and, most importantly, in human disease diagnostics and in phenotype prediction for genetic manipulation of plants and animals. Further, the integration of DNA chip and protein characterization data is an important step in correlating the results of genomic and protemic studies.
There exist several DNA chips which are used to conduct multiple sample parallel bioassays. For example, U.S. Pat. Nos. 5,800,992 and 5,744,305 to Fodor et al. discloses an oligonucleotide-based chip (herein called the “Fodor chip”) and U.S. Pat. No. 5,807,522 to Brown et al. discloses a cDNA-based microarray chip (herein called the “Brown chip”). Conventional biochips (the Fodor and Brown chips, and other two dimensional or surface-based biochips such as the Affymetrix type of biochip and the Stanford type of glass-slide microarray or Bio-Informatics Group's 3D biochip) are designed for parallel assays of a large number of bioelements (e.g. genes) for a single biosample (e.g. patients) or parallel assays of a single gene for a number of biosamples. For conventional biochips, data for different biosamples are usually generated at different times or under different experimental conditions. Experimental errors are the most common obstacles for the usefulness of the experiment because they prevent accurate comparison between the biosamples, which usually is the goal of the experiment.
The Fodor chip (U.S. Pat. Nos. 5,800,992 and 5,744,305) generally utilizes a flat silicon surface for in situ synthesis of the oligonucleotides on the chip surface using combinatory chemistry. The Fodor chip is typically limited to short oligonucleotide lengths, where the oligos have a small number (ie: 25) of nucleotide bases. The Fodor chip, therefore, may also be limited by experimental error associated with on-chip oligonucleotide synthesis and with short oligonucleotide hybridization error, which is generally associated with non-specific hybridization in a relaxed condition. Thus, due to these inherent experimental errors, techniques utilizing the Fodor chip may be prone to poor experimental repeatability. In addition, the Fodor chip may further be limited by slow hybridization rates due to the small effective hybridization area and random probe solution flow on the chip surface. In some instances, RNA amplification may also be required to increase the RNA concentration in the probe solution, which may make the Fodor chip unsuitable for certain applications. Thus, procedures involving the Fodor chip may be cost inefficient due to the complexities and limitations involved in producing the chip (labor intensive and time consuming), capturing the necessary images, and analyzing the collected data.
The Brown chip (U.S. Pat. No. 5,807,522) utilizes cDNA samples disposed in a microarray on the surface of a chip comprising a glass slide. The cDNA segments are typically chosen from cDNA libraries of EST sequencing projects. Each cDNA segment may range in length from several hundred to several thousand nucleotides. The nucleotide sequences in the cDNA segments are generally known, though cDNA segments without nucleotide sequence information and synthetic oligos may also be used in fabricating a Brown chip. The cDNA samples are usually delivered onto the chip using a robot having a three-dimensional motion control system and the ability to concurrently deposit multiple samples using a plurality of spotting pins. However, the Brown chip may also experience limitations such as, for example, error in the x-y positioning of the spotting pins by the robot and varying amounts of the cDNA samples deposited at each spot on the chip. In addition, hybridization error may be a limiting factor due to the small effective hybridization area on the chip and possibly due to secondary structure formed by single-strand oligonucleotides. Further, techniques using the Brown chip may be subject to extended hybridization times measured, for instance, in hours (for example, overnight hybridization). As with the Fodor chip, the Brown chip may also be difficult-to produce and may be limited in its practicality due to the limited surface area available on the chip.
Thus, the DNA samples reside on the surface of, for example, a glass slide or a silicon wafer according to both the Fodor and the Brown DNA chips. However, though the Fodor and Brown DNA chips are useful for some small-scale research in functional genomics, they are not suitable for future practical applications primarily due to high cost, time intensive fabrication of the DNA chip, and poor accuracy of experimental results. The poor accuracy of a surface-based biosample assay apparatus and method, for example, according to both Fodor and Brown, typically results from the low concentration of the complementary strands of DNA (or RNA) in the probe solution and the small effective hybridization area of the spots on surface-based chips. The biosamples are usually introduced to conventional biochips by hand or a robotic liquid handling instrument which is time consuming and subject to mistakes in the liquid handling. Further, the surface-based chips, such as the Brown chip, are often prepared using the robotic system for transferring biosamples from a mass solution to individual spots on a glass substrate to form the microarray. The biosample transfer may be accomplished, for example, by a robot operating at an overall rate of about four dots per second. Since a microarray may include, for instance, multiple thousands of individual samples, a microarray may be prone to lengthy formation times as well as possible contamination due to the robotic system.
The limited surface area and between sample variance for these conventional biochips points out a need in the art for a biochip based technology that can drastically increase the number of bioelements, the number of biosamples, or both being tested, and minimize between sample variances by allowing simultaneous testing of large numbers of biosamples.